A major problem in the treatment of cancer remains its early detection. Early detection enables therapeutic treatment from the onset of the disease resulting in successful treatment in many cases. Current methods for detection and quantitation of neoplastic cells employ a combination of techniques such as histochemical stains, exogenous labeling of surface markers, or light microscopy for morphological cell characterization for cancer cell recognition. These techniques are time consuming and are not very accurate, as a high number of false positive results can be obtained.
Raman microspectroscopy has been used for detection and identification in various scientific disciplines, including materials science, biology and medicine. G. Turrell and J. Corset, Raman microscopy, (Academic Press, London, 1996). Raman microspectroscopy uses a focused laser beam to illuminate particles, and the resulting scattered light is detected and analyzed. The incident light on the sample excites molecules in the sample to produce elastic scattering and inelastic scattering. The inelastically scattered light spectrum is called a Raman spectrum. The molecular composition and structure information of the particles can be obtained from positions, intensities, and line-widths of the Raman peaks in the spectra. One difficulty associated with Raman spectroscopy is the very low signal intensity which is inherent to Raman scattered light. It is well known that the scattered light intensity scales with the frequency raised to the fourth power. The weak Raman signal must be distinguished from Rayleigh scattered light, which is elastically scattered light of the same frequency as the incident light and which constitutes a much greater fraction of the total scattered light. The Raman signal can be separated from Rayleigh scattered light through the use of filters, gratings, or other wavelength separation devices, however, this can have the effect of further weakening the measured Raman signal through the additional attenuation which can occur when the light passes through a wavelength separation device.
Puppels demonstrated the use of confocal Raman microspectroscopy on a single cell (Puppels et al. (1990) Nature 347:301-303). The method has had many applications for biological studies, including the use of the technique as a potential diagnostic tool for cancer detection. Raman spectroscopy of tissue and cells, which can provide detailed molecular information about the DNA, protein, lipid, and carbohydrate content, has been suggested as a diagnostic tool for cancer detection. For example, Raman spectra studies of squamous dysplasia, a precursor to cervical cancer, classified high-grade dysplasia from all others based on the observed increases in the intensity ratios of 1454 cm−1 to 1656 cm−1 and decreases in the intensity ratio of 1330 cm−1 to 1454 cm−1 for the squamous dysplastic tissues. These peaks were assigned to collagen, phospholipids, and DNA. Tumor and normal bronchial tissue can be classified based on observed higher Raman signals from nucleic acids, typtophan, phenylalanine, and lower signals from phospholipids, proline, and valine in tumor tissue. Specifically, peaks ascribed to nucleic acid at 1223 cm−1 and 1335 cm−1 increase, peaks ascribed to collagen/phospholipid at 1302 cm−1 and 1405 cm−1 decrease, tryptophan and phenylalanine peaks such as 1618 cm−1 and 1602 cm−1, respectively, increase. Thus, Raman spectroscopy can be used to characterize diseases.
Optical trapping using a single laser beam, first pioneered by Ashkin et al. in 1986, utilizes single photons of light to impart radiation pressure forces on a particle. It has been shown that when a laser source is tightly focused, it is possible to generate both radiation axial scattering forces and transverse gradient forces to draw a particle towards the center of the beam into a stable trap at the laser focus. “Optical tweezers” have been used as a biological tool for the manipulation of single biological species, including manipulation of bacteria, viruses, single cells, subcellular organelles, single motor proteins, and single DNA molecules.
Recently, optical trapping combined with confocal Raman spectroscopy for simultaneously probing the molecular information of the trapped particle using a single laser source has attracted increased interest in the biophysical study of the molecular changes in the subcellular components of single live cells over an extended period of time. Confocal Raman microspectroscopy has been shown to provide information on the DNA, protein, and lipid constituents of a live biological cell with distinct bands assignable to nucleotide bases, amide, and others. The PCT publication, WO 2004/008121 by Li and Dinno, shows that an optical trap for cells combined with Raman spectroscopy can be used to distinguish between live yeast and microbial cells and unstained dead yeast and microbial cells. U.S. Pat. No. 6,067,859 to Kas et al. discloses an “optical stretcher” where a tunable laser is used to trap and deform cells between two counter-propagating beams generated by a laser. The system is utilized to detect single malignant cancer cells.
The combination of optical trapping with Raman spectroscopy has the advantages of 1) enabling the study of micro and nano particles in solution over an extended period of time that might otherwise not be able to be probed due to their constant Brownian motion in solution, 2) maximizing Raman signal collection, and 3) probing of natural suspension of cells.
There is a need for diagnostic methods and apparatus involving a single rapid technique that can accurately and non-invasively identify and sort single cancerous cells, such as leukemic cells, from healthy cells for the diagnosis and treatment of cancers in a clinical setting.